Mitochondrial Measurements

Alamar Blue

10 mL media
1 mL Alamar Blue (found in left normal Freezer; changes color of media to red/pink)

1. Mix Alamar Blue and Media together in a trough.
2. Aspirate old media from 96-well plate.
3. Add 100ul of Alamar Blue/Media Solution to each well.
4. Incubate for 4 hours.
5. Read on plate reader
   
   1. Ensure the bulb is warmed up
   2. Click on Re-read
   3. Save and Export results to Excel
   4. Title with Date, Initial and MTT.

MitoTracker

10 mL media
2.5 ul Mitotracker (found in -20 freezer, top shelf, long white box; KEEP IN DARK)

• Make sure it is completely thawed before using
• Add 93 ul dMSO (on dry Chemical Shelf in dark bottle) if the Mitotracker isn’t purple

1. Mix media and Mitotracker really well
2. Aspirate off old media from 96- well plate
3. Add 100 ul of Mitotracker Media toe ach well
4. Incubate for 15 minutes in the dark
5. After incubation:
   
   1. Aspirate old media
   2. Wash each well with 100ul PBS
      
      1. Place PBS on the side of the well and expel gently so as to not blow the cells off the plate
   3. Aspirate off PBS
   4. Add 100ul new PBS to each well
   5. Use the Plate Reader for Mitotracker to read
      
      1. Ensure the bulb is warmed up
      2. Click on Re-read
      3. Save and Export results to Excel
      4. Title with Date, Initial and Mitotracker

MTT

Preparation

1. Prepare a 12mM MTT stock solution by adding:
   
   1. 1mL of sterile PBS to 5mg MTT
   2. Mix by vortexing or sonication until dissolved
   3. Once prepared, the MTT solution can be stores for 4 weeks at 4 degrees Celsius, protected from light
2. Prepare SDS_HCL solution
   
   1. Add 10mL of 0.01M HCL to 1g of SDS.
   2. Mix the solution gently by inversion or sonication until the SDS dissolves.
   3. Once prepared, the solution should be used promptly.

Treatment

1. Mix 1mL 12mM MTT with 10mL media
2. Aspirate old media from plate
3. Add 100ul per well of the MTT/Media solution to each well of a 96-well plate.
4. Incubate for 4 hours.
5. Add 100ul of the SDS-HCL solution to each well and mix thoroughly.
6. Incubate the plate for 4 hours. Longer incubation will decrease the sensitivity of the assay.
7. Mix each sample again using a pipette.
8. Use the Plate reader and read absorbance at 570nm.
   
   1. Ensure the bulb is warmed up
   2. Click on Re-read
   3. Save and Export results to Excel
   4. Title with Date, Initial and MTT.